KRT10 Recombinant Monoclonal Antibody

What is KRT10 and why is it significant in research?

KRT10 (Cytokeratin 10) is a 56.5-58.8 kDa protein that serves as a key structural component of the epidermis. It plays a central role in maintaining the structural integrity of the epidermis, contributing to the formation of a protective skin barrier, and facilitating epidermal differentiation and cornification processes. The protein has 584 amino acid residues in humans and is localized primarily in the cytoplasm, though it is also secreted. KRT10 is a member of the Intermediate filament protein family and is particularly significant in research because its expression strictly parallels the extent of differentiation in the epidermis. It is absent in the basal layer, appears in the first suprabasal layers, and increases in concentration toward the granular layer. Mutations in the KRT10 gene are associated with various skin disorders characterized by skin fragility and abnormalities, making it an important target for dermatological research .

How are KRT10 recombinant monoclonal antibodies produced?

KRT10 recombinant monoclonal antibody production is a meticulously coordinated process that begins with in vitro cloning. The KRT10 antibody genes are first incorporated into expression vectors, which are then introduced into host cells. This facilitates the expression of the recombinant antibody within a cell culture environment. Following expression, the antibody undergoes purification from the supernatant of transfected host cell lines through an affinity-chromatography purification method. This approach ensures high specificity and consistency in the final antibody product. Some specific monoclonal antibodies, such as RKSE60, are derived by fusion of SP2/0 mouse myeloma cells with spleen cells from mice immunized with cytokeratins from human epidermis, representing a more traditional hybridoma approach that may be used alongside recombinant technologies .

What tissue expression pattern should researchers expect when using KRT10 antibodies?

When using KRT10 antibodies, researchers should expect a distinctive expression pattern that reflects the protein's role in epidermal differentiation. KRT10 is predominantly expressed in keratinizing stratified epithelia and in differentiated areas of highly differentiated squamous cell carcinomas. In normal skin, KRT10 is absent in the basal layer of the epidermis but appears in the first suprabasal layers and increases in concentration toward the granular layer. This expression pattern strictly parallels the extent of differentiation in the epidermis. KRT10 serves as a marker for Granular Keratinocytes, Spinous Keratinocytes, and Onychocyte Keratinocytes. When conducting immunohistochemistry, researchers should anticipate positive staining in the suprabasal layers of the epidermis with a cytoplasmic localization pattern. The absence of staining in the basal layer provides an internal negative control in skin samples, which can help validate staining specificity .

What are the optimal applications for KRT10 recombinant monoclonal antibodies?

KRT10 recombinant monoclonal antibodies demonstrate versatility across multiple research applications. Immunohistochemistry (IHC) is the most widely used application, with these antibodies performing exceptionally well on both formalin-fixed paraffin-embedded (FFPE) tissues and frozen sections at dilutions typically ranging from 1:50-1:200. Western blotting is also a common application, with recommended dilutions of 1:100-1:1000, allowing researchers to detect the 56.5-58.8 kDa KRT10 protein. Flow cytometry applications typically employ dilutions of 1:100-1:200, enabling the identification and sorting of keratinocyte populations at different stages of differentiation. Additionally, these antibodies are suitable for immunocytochemistry and immunofluorescence studies, providing insights into KRT10 subcellular localization. When selecting an application, researchers should consider that over 1,300 citations in the literature describe the use of KRT10 antibodies, with IHC being the predominant methodology, suggesting its reliability for detecting this protein in tissue contexts .

How should researchers optimize KRT10 antibody protocols for immunohistochemistry?

When optimizing KRT10 antibody protocols for immunohistochemistry, researchers should implement a systematic approach to achieve optimal staining. For formalin-fixed, paraffin-embedded tissues, antigen retrieval is critical and typically requires boiling tissue sections in 10mM citrate buffer (pH 6.0) for 10-20 minutes, followed by cooling at room temperature for 20 minutes. Optimal antibody dilutions generally range from 1:50-1:200, but this should be determined through titration experiments for each specific antibody clone and tissue type. Incubation times of 30 minutes at room temperature are typically effective, though overnight incubations at 4°C may enhance sensitivity for weakly expressed targets. The detection system significantly impacts results, with the avidin-biotinylated horseradish peroxidase complex (ABC) method generally providing excellent signal-to-noise ratios. Positive controls should include tissues known to express KRT10, such as esophagus or tonsil, while negative controls should omit the primary antibody. For antibodies that recognize multiple species, researchers should verify cross-reactivity using appropriate tissue samples from each target species. Finally, counterstaining with hematoxylin provides cellular context without obscuring the DAB or other chromogenic signals .

What considerations are important when using KRT10 antibodies for Western blotting?

When using KRT10 antibodies for Western blotting, researchers should consider several critical factors to ensure optimal results. Sample preparation is crucial—for epidermis or skin samples, care must be taken to efficiently extract intermediate filament proteins, which may require specialized lysis buffers containing urea or high salt concentrations. Running conditions should be optimized to resolve the 56.5-58.8 kDa KRT10 protein effectively, with 10% SDS-PAGE gels typically providing good separation. Transfer conditions may need optimization, as intermediate filament proteins sometimes require extended transfer times or modified buffer compositions. Blocking solutions should be carefully selected, with 5% non-fat dry milk often providing better results than BSA for these antibodies. Dilution ranges typically fall between 1:100-1:1000, but researchers should perform titration experiments to determine optimal conditions for their specific antibody. Given KRT10's expression in differentiated keratinocytes, positive controls such as A431, HeLa, or MCF7 cell lysates can be used to validate antibody performance. When analyzing results, researchers should be attentive to potential post-translational modifications or proteolytic cleavage products that might affect the observed molecular weight. Finally, stripping and reprobing with antibodies against housekeeping proteins should be performed to ensure equal loading across samples .

How can researchers distinguish between true KRT10 signals and potential cross-reactivity with other keratins?

Distinguishing between true KRT10 signals and potential cross-reactivity with other keratins requires a multi-faceted validation approach. Researchers should first select highly specific monoclonal antibodies, such as clone RKSE60, which has been demonstrated to react exclusively with cytokeratin 10 in keratinizing stratified epithelia. Parallel experiments with antibodies targeting other keratins (particularly KRT1, KRT2, and other epithelial keratins) can help establish staining pattern differences. Examining the tissue distribution pattern is particularly informative—authentic KRT10 staining should be absent in the basal layer, appear in the first suprabasal layers, and increase toward the granular layer of the epidermis. This distinctive gradient serves as an internal validation feature. Knockout or knockdown experiments provide definitive confirmation, as the signal should disappear or diminish in KRT10-depleted samples. Sequential immunoprecipitation can also be employed to first deplete samples of other keratins before probing for KRT10. For mass spectrometry-based validation, researchers can perform immunoprecipitation followed by peptide identification to confirm antibody specificity. Finally, comparing results across different antibody clones that recognize distinct epitopes of KRT10 can help validate findings, as consistent results across multiple antibodies strongly suggest specific KRT10 detection rather than cross-reactivity .

What experimental design considerations are essential when investigating KRT10 expression in pathological skin conditions?

When investigating KRT10 expression in pathological skin conditions, researchers must implement a comprehensive experimental design that accounts for multiple variables affecting keratin expression. Case-control matching is paramount—control samples should be matched for anatomical location, age, gender, and sun exposure, as these factors influence baseline KRT10 expression. Sampling methodology should include multiple biopsies from different regions of the lesion (center, periphery, and transition zones) to capture expression heterogeneity. For conditions like squamous cell carcinomas, stratification by tumor size and clinical stage is essential, as KRT10 expression patterns vary significantly between early-stage (< 2 cm, clinical stage I) and advanced tumors (> 2 cm, clinical stages II and III). Co-staining with proliferation markers (Ki67) and differentiation markers (involucrin, loricrin) provides contextual information about the relationship between KRT10 expression and cellular state. Quantification methods should incorporate both intensity and distribution parameters—the percentage of positive cells, staining intensity, and spatial distribution within tissue layers. Longitudinal sampling for progressive conditions can reveal temporal changes in KRT10 expression patterns. Finally, correlative analysis with clinical parameters (disease severity, treatment response, recurrence rates) enhances the translational relevance of findings. This comprehensive approach enables researchers to distinguish disease-specific alterations in KRT10 expression from normal biological variation .

How can researchers effectively use KRT10 antibodies in flow cytometry for keratinocyte differentiation studies?

Effective use of KRT10 antibodies in flow cytometry for keratinocyte differentiation studies requires careful optimization of multiple parameters to achieve reliable results. Sample preparation is critical—trypsinization conditions must be gentle to preserve keratin epitopes, with researchers often benefiting from brief enzymatic treatment followed by mechanical dissociation. Since KRT10 is an intracellular protein, permeabilization is essential, with methanol/acetone fixation often yielding better results than formaldehyde-based protocols for keratin detection. Antibody concentration should be determined through titration experiments, typically starting at 0.5-1μg per million cells in 0.1ml volume. Multi-parameter analysis significantly enhances the value of these studies—co-staining with basal keratinocyte markers (K5/K14), proliferation markers (Ki67), and other differentiation markers (involucrin, filaggrin) allows for the identification of discrete keratinocyte subpopulations at different maturation stages. Compensation controls are particularly important when using multiple fluorophores, as keratinocytes exhibit relatively high autofluorescence. Gating strategies should first exclude dead cells and debris, followed by selection of single cells, then analysis of KRT10-positive populations in relation to other markers. For quantitative studies, calibration beads should be used to standardize fluorescence intensity values across experiments. This methodical approach enables researchers to accurately track keratinocyte differentiation stages in normal development, disease states, or in response to experimental treatments .

How does KRT10 function interact with other epidermal proteins in maintaining skin barrier integrity?

KRT10 functions within a complex network of structural and regulatory proteins to maintain skin barrier integrity. As a type I keratin, KRT10 preferentially forms heterodimers with the type II keratin KRT1, creating intermediate filaments that form the cytoskeletal framework of suprabasal keratinocytes. This KRT1/KRT10 network provides mechanical resilience to the epidermis, distributing mechanical forces and preventing cell fragility. Beyond its structural role, KRT10 interacts with desmosomes through desmoplakin and other desmosomal proteins, creating a transcellular network that strengthens cell-cell adhesion. KRT10 also participates in regulatory functions—it can sequester and modulate the activity of signaling molecules such as Akt and PKCζ, thereby influencing differentiation and proliferation pathways. During terminal differentiation, KRT10 interacts with filaggrin and loricrin as these proteins become incorporated into the cornified envelope, contributing to the formation of a tightly cross-linked protein-lipid matrix that serves as the primary permeability barrier. The organization of KRT10 filaments also influences the trafficking and secretion of lamellar bodies, which deliver lipids and enzymes crucial for barrier function to the extracellular space. Mutations in KRT10 disrupt these interactions, leading to cytoskeletal collapse, impaired cell-cell adhesion, abnormal differentiation, and ultimately compromised barrier function manifesting as conditions like epidermolytic hyperkeratosis. Understanding these interactions provides mechanistic insights into how KRT10 contributes to epidermal homeostasis and offers potential targets for therapeutic intervention in skin disorders .

What methodological approaches can resolve contradictory data regarding KRT10 expression in different experimental systems?

Resolving contradictory data regarding KRT10 expression across different experimental systems requires a systematic methodological approach that addresses potential sources of variability. First, researchers should standardize detection methods—comparing antibody-based techniques (immunohistochemistry, immunofluorescence, Western blotting) with transcript-based approaches (qRT-PCR, RNA-seq, in situ hybridization) can distinguish between transcriptional regulation and post-transcriptional mechanisms affecting KRT10 expression. Antibody validation is critical—comparing multiple antibody clones that recognize different epitopes helps exclude clone-specific artifacts, while validation in knockout/knockdown systems confirms specificity. Culture conditions substantially impact keratinocyte differentiation—calcium concentration, serum factors, confluence level, and air-liquid interface exposure dramatically affect KRT10 expression, so standardizing these parameters is essential. In 3D skin models, matrix composition, culture duration, and media formulation must be precisely controlled and reported. For in vivo studies, controlling for anatomical location, age, gender, and environmental factors (particularly UV exposure) minimizes biological variability that might be misinterpreted as experimental inconsistency. Statistical approaches such as meta-analysis of published data sets and multivariate analysis help identify factors consistently associated with KRT10 expression across diverse experimental systems. Finally, time-course experiments often resolve apparent contradictions by revealing dynamic expression changes rather than static differences. This comprehensive approach not only resolves contradictions but often yields deeper insights into the biological regulation of KRT10 expression in different contexts .

What controls are essential when validating a new KRT10 recombinant monoclonal antibody for research use?

Validating a new KRT10 recombinant monoclonal antibody requires a comprehensive set of controls to ensure specificity, sensitivity, and reproducibility. Positive tissue controls are fundamental—normal human epidermis should show the characteristic pattern of KRT10 expression (absent in basal layer, present in suprabasal layers with increasing intensity toward the granular layer). Cell line controls should include both positive (A431, HeLa, MCF7) and negative (basal cell-derived lines) examples. Peptide blocking experiments, where the antibody is pre-incubated with the immunogen peptide before staining, should abolish specific staining if the antibody is truly specific. Cross-reactivity assessment across multiple species requires parallel staining of tissues from each claimed reactive species (human, mouse, rat, dog, zebrafish) to confirm conserved staining patterns. Technical negative controls must include primary antibody omission and isotype controls (using non-specific mouse IgG1 for a mouse monoclonal antibody). Molecular weight verification by Western blotting should demonstrate a band at the expected 56.5-58.8 kDa size. Comparison with established KRT10 antibody clones (such as RKSE60) provides benchmark validation, with similar staining patterns suggesting authentic KRT10 detection. For ultimate validation, KRT10 knockout or knockdown samples should show absent or reduced staining. Finally, orthogonal method validation, comparing protein detection with mRNA expression by in situ hybridization, provides confirmation of expression patterns. This systematic approach ensures that new KRT10 recombinant monoclonal antibodies meet the rigorous standards required for research applications .

How can researchers differentiate between native KRT10 and degradation products in experimental samples?

Differentiating between native KRT10 and its degradation products requires multiple analytical approaches that assess protein integrity. Western blotting with gradient gels (4-20%) can resolve the full-length 56.5-58.8 kDa KRT10 protein from smaller fragments, with researchers looking for distinct bands below the expected molecular weight that may represent degradation products. Domain-specific antibodies targeting different epitopes along the KRT10 protein provide critical insights—comparing staining patterns between antibodies recognizing N-terminal, internal, and C-terminal regions can reveal partial degradation when signals are discordant. Sample preparation significantly impacts keratin integrity—researchers should compare fresh samples with those subjected to different storage conditions (freeze-thaw cycles, extended storage) to assess stability. Protease inhibitor cocktails should be systematically included or omitted during extraction to evaluate their necessity for preserving intact KRT10. Two-dimensional gel electrophoresis followed by Western blotting or mass spectrometry can distinguish between degradation fragments and post-translationally modified forms of KRT10. For tissue sections, comparing the subcellular localization pattern between fresh and stored samples helps identify artifactual staining that may result from degradation. Time-course experiments examining samples collected at different intervals after tissue harvesting provide insights into the degradation kinetics of KRT10. Finally, recombinant KRT10 can serve as a degradation control when subjected to controlled proteolysis and analyzed in parallel with experimental samples. These methodological approaches allow researchers to confidently distinguish biologically relevant KRT10 signals from artifacts arising from protein degradation .

What experimental design strategies can determine whether changes in KRT10 expression are causative or consequential in disease processes?

Determining whether changes in KRT10 expression are causative or consequential in disease processes requires sophisticated experimental design strategies that establish causality rather than mere correlation. Genetic manipulation approaches provide the strongest evidence—introducing KRT10 mutations into normal keratinocytes should recapitulate disease phenotypes if alterations are causative, while correcting mutations in patient-derived cells should restore normal function. Temporal expression analysis in disease development is crucial—if KRT10 alterations precede other disease manifestations, this suggests a causative role, whereas changes that follow other pathological events likely represent consequences. Inducible expression systems allow precise temporal control of KRT10 expression or suppression, enabling researchers to determine exactly when KRT10 changes can influence disease trajectory. Dose-response relationships between KRT10 expression levels and disease severity provide evidence for causality—a consistent, quantitative relationship suggests direct involvement. Intervention studies using antisense oligonucleotides, siRNA, or CRISPR-based approaches to modulate KRT10 expression in disease models can establish whether normalizing expression ameliorates pathology. Pathway analysis combining KRT10 manipulation with inhibitors of hypothesized downstream effectors can map the mechanistic chain of events linking KRT10 to disease manifestations. Animal models with tissue-specific and inducible KRT10 modifications allow in vivo validation of hypotheses generated in cell culture systems. Finally, human genetic studies examining whether KRT10 variants segregate with disease in families or associate with disease risk in populations provide complementary evidence from a different analytical perspective. This multi-faceted approach enables researchers to confidently establish the place of KRT10 alterations in the causal chain of disease pathogenesis .

What are the comparative specifications of different KRT10 antibody clones?

This comparative table highlights the diversity of available KRT10 antibody clones, each with specific characteristics that make them suitable for particular research applications. Researchers should select the appropriate clone based on their experimental requirements, target species, and preferred application methodology .

What tissue expression levels of KRT10 should researchers expect across different epithelial tissues?

This expression table provides researchers with baseline expectations for KRT10 detection across various tissues. Understanding the normal expression pattern is crucial for interpreting pathological changes. The distinctive suprabasal-only pattern in stratified epithelia serves as an internal control for staining specificity, as the absence of staining in basal layers confirms antibody selectivity .

What methodological parameters affect the detection sensitivity of KRT10 in different experimental systems?

This methodological parameter table provides researchers with a systematic approach to optimizing KRT10 detection across different experimental platforms. The table highlights critical parameters that affect sensitivity and specificity, along with strategies for optimization and enhancement. By addressing these methodological variables, researchers can achieve consistent and reliable detection of KRT10, facilitating cross-platform validation of results .

How can KRT10 antibodies be integrated into single-cell analysis workflows for skin research?

KRT10 antibodies can be strategically integrated into single-cell analysis workflows to provide crucial insights into keratinocyte differentiation states at unprecedented resolution. For single-cell cytometry approaches, researchers should incorporate KRT10 antibodies in multi-parameter panels alongside markers for stemness (integrin β1, CD71), proliferation (Ki67), other differentiation stages (K14, involucrin, loricrin), and cell cycle status. Index sorting, where flow cytometry data are retained for each individually sorted cell, allows correlation of KRT10 expression levels with subsequent single-cell transcriptomics or other molecular analyses. For mass cytometry (CyTOF), KRT10 antibodies can be metal-conjugated and incorporated into panels of 30+ markers, enabling comprehensive phenotyping of epidermal populations with minimal spectral overlap concerns. In imaging mass cytometry or multiparameter immunofluorescence, KRT10 serves as a spatial reference for differentiation status, allowing researchers to map molecular heterogeneity within the architectural context of the tissue. For single-cell RNA sequencing integration, protein detection with KRT10 antibodies through CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) enables direct correlation between protein expression and transcriptional profiles at single-cell resolution, helping resolve post-transcriptional regulation mechanisms. Computational integration of KRT10 antibody staining with scRNA-seq using algorithms like Seurat or LIGER can align protein-defined populations with transcriptional states. This multimodal approach is particularly valuable for studying rare transitional states during differentiation or disease processes, revealing cellular heterogeneity that might be masked in bulk analyses .

What are the methodological considerations for studying KRT10 post-translational modifications in skin disease models?

Studying KRT10 post-translational modifications (PTMs) in skin disease models requires specialized methodological approaches to detect and characterize these often subtle but functionally significant alterations. Sample preparation is critical—flash freezing tissues and utilizing protease inhibitor cocktails supplemented with specific PTM inhibitors (phosphatase, acetylase, and ubiquitin-specific inhibitors) preserves modification states. Enrichment strategies significantly enhance detection sensitivity—phosphorylated KRT10 can be enriched using titanium dioxide or immobilized metal affinity chromatography, while ubiquitinated forms can be isolated using tandem ubiquitin binding entities. For immunodetection, modification-specific antibodies that recognize phosphorylated, ubiquitinated, SUMOylated, or acetylated KRT10 provide direct visualization, though these require rigorous validation using site-directed mutagenesis of modification sites. Mass spectrometry approaches offer comprehensive PTM mapping—a combination of bottom-up (tryptic peptides) and middle-down (larger fragments) proteomics provides complementary coverage of the KRT10 sequence. Multiple fragmentation methods (CID, ETD, HCD) should be employed, as certain modifications are better preserved with specific techniques. Quantitative analysis requires careful experimental design—using SILAC, TMT, or label-free quantification to compare PTM profiles between normal and disease states, with appropriate normalization to total KRT10 levels. In situ proximity ligation assays can detect specific KRT10 PTMs within tissue architectural context by using antibody pairs targeting both KRT10 and the modification. For functional validation, site-directed mutagenesis of modification sites followed by expression in keratinocyte models helps establish causality between specific PTMs and altered KRT10 properties. This integrative approach enables researchers to comprehensively characterize KRT10 PTM landscapes in skin disease models, providing mechanistic insights into pathogenesis .

What potential exists for using KRT10 as a biomarker in liquid biopsy approaches for epithelial disorders?

KRT10's potential as a biomarker in liquid biopsy approaches for epithelial disorders stems from its tissue-specific expression pattern and release during pathological processes. The methodological foundation begins with optimized sample collection—protocols for serum, plasma, and interstitial fluid should be standardized with controlled pre-analytical variables (collection tubes, processing times, centrifugation parameters) to ensure reproducibility. Detection methods vary in sensitivity—traditional ELISA has a detection limit around 0.1-1 ng/ml, while digital ELISA platforms can achieve femtomolar sensitivity, crucial for detecting the relatively low KRT10 concentrations in circulation. Mass spectrometry-based approaches offer advantages in specificity by detecting signature peptides unique to KRT10, distinguishing it from other keratins. For circulating tumor cell (CTC) analysis, KRT10 antibodies can be incorporated into positive selection strategies for cells of epithelial origin, particularly for squamous cell carcinomas. In exosome research, KRT10 detection in isolated exosomes may provide insights into the differentiation status of the originating cells. Clinical validation requires careful study design—longitudinal sampling with defined time points relative to disease progression, treatment initiation, and clinical outcomes. Reference ranges must be established across diverse demographics, accounting for age, gender, ethnicity, and anatomical variation in normal KRT10 expression. Multiplexing KRT10 with other biomarkers significantly enhances diagnostic value—combining with inflammatory markers, other keratins, or disease-specific molecules can improve sensitivity and specificity. Importantly, normalization strategies are essential—relating KRT10 levels to total protein, albumin, or using ratio-based approaches with other keratins helps control for pre-analytical variables. This methodologically rigorous approach positions KRT10 as a promising biomarker candidate, particularly for disorders affecting keratinizing epithelia .